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Real-time tracking of bacterial membrane vesicles reveals enhanced membrane traffic upon antibiotic exposure.

ABSTRACT: Membrane vesicles are ubiquitous carriers of molecular information. A broad understanding of the biological functions of membrane vesicles in bacteria remains elusive because of the imaging challenges during real-time in vivo experiments. Here, we provide a quantitative analysis of the motion of individual vesicles in living microbes using fluorescence microscopy, and we show that while vesicle free diffusion in the intercellular space is rare, vesicles mostly disperse along the bacterial surfaces. Most remarkably, when bacteria are challenged with low doses of antibiotics, vesicle production and traffic, quantified by instantaneous vesicle speeds and total traveled distance per unit time, are significantly enhanced. Furthermore, the enhanced vesicle movement is independent of cell clustering properties but rather is associated with a reduction of the density of surface appendages in response to antibiotics. Together, our results provide insights into the emerging field of spatial organization and dynamics of membrane vesicles in microcolonies.

SUBMITTER: Bos J

PROVIDER: S-EPMC7817102 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Real-time tracking of bacterial membrane vesicles reveals enhanced membrane traffic upon antibiotic exposure.

Bos Julia J Cisneros Luis H LH Mazel Didier D

Science advances 20210120 4

Membrane vesicles are ubiquitous carriers of molecular information. A broad understanding of the biological functions of membrane vesicles in bacteria remains elusive because of the imaging challenges during real-time in vivo experiments. Here, we provide a quantitative analysis of the motion of individual vesicles in living microbes using fluorescence microscopy, and we show that while vesicle free diffusion in the intercellular space is rare, vesicles mostly disperse along the bacterial surfac ...[more]

PMID: 33523924

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Project description:The nucleoid-associated proteins Hha and YdgT repress the expression of the toxin ?-hemolysin. An Escherichia coli mutant lacking these proteins overexpresses the toxin ?-hemolysin encoded in the multicopy recombinant plasmid pANN202-312R. Unexpectedly, we could observe that this mutant generated clones that no further produced hemolysin (Hly(-)). Generation of Hly(-) clones was dependent upon the presence in the culture medium of the antibiotic kanamycin (km), a marker of the hha allele (hha::Tn5). Detailed analysis of different Hly(-) clones evidenced that recombination between partial IS91 sequences that flank the hly operon had occurred. A fluctuation test evidenced that the presence of km in the culture medium was underlying the generation of these clones. A decrease of the km concentration from 25 mg/l to 12.5 mg/l abolished the appearance of Hly(-) derivatives. We considered as a working hypothesis that, when producing high levels of the toxin (combination of the hha ydgT mutations with the presence of the multicopy hemolytic plasmid pANN202-312R), the concentration of km of 25 mg/l resulted subinhibitory and stimulated the recombination between adjacent IS91 flanking sequences. To further test this hypothesis, we analyzed the effect of subinhibitory km concentrations in the wild type E. coli strain MG1655 harboring the parental low copy number plasmid pHly152. At a km concentration of 5 mg/l, subinhibitory for strain MG1655 (pHly152), generation of Hly(-) clones could be readily detected. Similar results were also obtained when, instead of km, ampicillin was used. IS91 is flanking several virulence determinants in different enteric bacterial pathogenic strains from E. coli and Shigella. The results presented here evidence that stress generated by exposure to subinhibitory antibiotic concentrations may result in rearrangements of the bacterial genome. Whereas some of these rearrangements may be deleterious, others may generate genotypes with increased virulence, which may resume infection.

Dataset Information

Real-time tracking of bacterial membrane vesicles reveals enhanced membrane traffic upon antibiotic exposure.

Publications

Real-time tracking of bacterial membrane vesicles reveals enhanced membrane traffic upon antibiotic exposure.

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